제4절 결 과
4.1 Reads output of genomicDNA of the 10 samples
10개의 잎 샘플 개체에서 각각 gDNA를 추출 후 Raw data를
4.4 SNPs mapping on transcripts data
N/T그룹 내에서 모든 샘플이 일치하며 그룹 간의 서로 다른 homo/homo SNPs로 최종으로 92개의 SNPs를 선발하였다. 이 SNPs가 존재하는 위치의 reference genome sequence와 제 2 장에서 생산한 transcripts의 sequence를 mapping하여 선발한 92개의 SNPs가
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위치한 서열이 기능을 하는 위치인지 알아보고자 하였다. 그 결과 총 29개의 transcripts의 sequence에 mapping이 되었으며, NCBI의 data base로 annotation한 결과 19개의 SNPs의 위치는 Pinus taeda clone의 complete sequence에 mapping되었으며, 이 부분의 기능은 아직 밝혀져 있지 않다. 그 외 Picea sitchensis clone은 2개, Picea glauca clone에 3개의 SNPs가 mapping 되었다. 그리고 Pinus contorta에서 voucher DSG655 plastid, complete genome (MH612863.1)에 mapping 되었다. SNPs 위치의 기능이 밝혀져 있는 transcripts은 Pinus pinaster의 malate dehydrogenase (mdhB) gene, partial cds(HM853297.1)였다(Table 3-4). mdh는 말산을 탈수소하며, 옥살로아세트산을 생성하는 반응을 촉매하는 효소로 자연계에 널리 분포하고 있는 효소이다. 앞서 언급한 반응은 에너지원인 ATP를 생산하는 TCA회로의 점화제와 글루코스 재합성의 준비단계로서 중요하다. Pinus pinaster의 transposon PpRT1, complete sequence (DQ394069.1)가 mapping 되었다(Rocheta et al., 2007). PpRT1은 5,966 bp이며, Pinus radiata의 IFG7 gypsy 레트로 트랜스포존과 밀접하게 관련되어있다. 긴 말단 반복 (LTRs)은 각각 333bp를 가지며, 이들 사이에 5.4 %의 서열 차이를 나타낸다. 특이적인 polypurine tract (PPT)와 primer binding site (PBS) 외에, PpRT1은 retroviral gene gag와 pol과 상동성이 있는 internal region을 가지고있다. PpRT1은 IFG7 gypsy 와의 서열 차이가 있어 Pinus radiata내에서 잘 보존된 retrotransposon인데 반송에서도 발견 된 것으로 보아 Pinus 속에서 잘 보존된 orthologous retrotransposon인 것으로 판단된다.
85 Table 3-1. Data output from 10 samples
Sample ID Total read bases (bp) total reads GC (%) AT (%) Q20 (%) Q30 (%)
T1 107,627,971,020 712,768,020 38.499 61.5 98.032 94.331
T4 134,933,354,172 893,598,372 38.584 61.42 97.69 93.933
T5 142,086,574,794 940,970,694 38.52 61.48 97.691 93.42
T9 152,804,097,566 1,011,947,666 38.399 61.6 97.704 93.388
T12 132,898,763,260 880,124,260 38.526 61.47 97.714 93.409
N2 135,032,557,246 894,255,346 38.539 61.46 97.504 93.095
N15 129,806,754,984 859,647,384 38.478 61.52 96.858 91.577
N19 122,937,111,456 814,153,056 38.292 61.71 97.65 93.299
N27 127,679,873,778 845,562,078 38.479 61.352 97.504 92.922
N37 139,214,412,854 921,949,754 38.4 61.6 97.323 92.648
Average 132,502,147,113 877,497,663 38.47 61.51 97.57 93.20
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Figure 3-2. Average depth per each samples. Mapping program : BWA v 0.7.17 with ‘mem’ engine
Table 3-3. Selection of different SNPs of N/T group
Type (N/T) SNP number
Homo/Homo 7,356
Homo/Hetero 4,869
Hetero/Homo 4,441
Total 16,666
87 transposon PpRT1, complete sequence(DQ394069.1)이었다.
PpRT1에 대한 연구에서 보면 PpRT1은 Pinus radiata내에서 잘
DNA demethylation, heterochromatin 탈증축 그리고 transposon 활성화가 일어나며(He et al., 2019), 애기장대 연구에서는 동반
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요소가 개화에 관여할 가능성이 있는 것으로 판단하였다.
본 연구를 시작하기 앞선 관찰에서 풍매차대집단의 개체들에서 암구화의 발달 위치를 관찰하여 다년간의 일관된 개화 위치에 대한 경향성이 존재한다고 판단하였다. 그리고 이 현상이 지속된다면 암구화가 정단에만 발달하는 개체들과 측면에만 발달하는 개체간 유전적인 차이가 있을 것이라는 가설을 세웠었다.
연구 결과 두 그룹간에 차이를 보이는 SNP들을 다수 찾을 수 있었느며 그룹 내에서 일치하며 그룹 간 차이가 나는 homo/homo SNPs를 92개 찾아내었다. 이는 전사수준에서 변하는 것이 아닌 유전적인 요인으로 인한 화기의 위치가 바뀌는 것이라는 가설을 뒤받침 해줄 수 있는 일부 근거가 될 수 있으며, 초기에 세웠던 가설을 기각하지 않는다는 것을 증명하였다.
향후 실제로 SNPs의 위치를 반송 내의 유전체 서열과 염색체 내에서 파악하여, 변이가 존재하는 원인을 증명하고 SNPs의 주변부를 더욱 탐색하여 주변 유전자에 SNPs가 어떠한 영향을 끼치는가에 대한 연구를 지속적으로 해나가야 할 것이다.
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Abstract
Pinus densiflora f. multicaulis Uyeki is one of the unique varieties of pine trees and is distributed mainly in Korea and Japan. Unlike the pine tree, the trunk is not developed straightly, but several branches are separated from the beginning of the ground. Thus, various types of tree appear for each individual, and they have excellent value. P. densiflora f. multicaulis are rarely distributed in nature and propagate by combining them for commercial or research purposes. In addition, Pinus species have been reported to have bisexual strobilus mixed male and female organ, and the development of bisexual strobilus has been observed more frequently in other species than in other species. and recently some transcriptome analysis has been conducted in two overseas species. Also, studies on the reasons for the development of bisexual strobili in the pine trees and the factors determining the reproductive organs have been continuously carried out. In Korea, the genome sequencing project of Pinus densiflora is in the beginning stage, and genetic research on P. densiflora f. multicaulis is also in the basic stage. In this study, we selected P. densiflora f.
multicaulis Uyeki that have a low height and a lot of strobili, and analyzed the characteristics of the strobili and mature cones of P.
densiflora f. multicaulis by the morphological, transcriptomical and genetical variances differences. times and growth than the cones and seeds developed at the apical them. However, it was confirmed that the seeds obtained from the side cones were close to the germination rate of the seeds obtained from the apical cones.
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As a result of analysis of transcriptome, the LEA gene family, cyplp029 gene, WRKY transcription factor ZmWRKY17, and dhn_ESK2 and so on were expressed in male strobili. In the beginning of biosexual strobili, we identified PtLTP1 to control ethylene, PIN1 to transport Auxin and NPF7 which transports nitrogen respectively. In female strobili, gibberellin, which is always expressed specifically in female tissues, the precursors of hormones such as CYPs and gibberellin metabolism genes, which are involved in the biosynthesis of gibberellin and brassinosteroid, in addition, in female strobili of side part, LINE-1 retrotransposable element was identified.
As a result of the SNPs comparative analysis of normal population only developed apical cones and abnormal population developed side cones, only 16,666 SNPs were found in the two groups. Homo / homo SNPs 92, all of which matched within the population and differ between groups, were selected. Mapping to the sequence and RNA-seq data of the reference genome at the selected SNPs site revealed the transposon PpRT1 of Pinus pinaster. The function of the sequence of the selected SNPs should be clarified through further studies.
From the results of this study, it was found that the same cone as the apical of branch is also developed in the lateral of branches. And genes that are presumed to be expressed by the flower developmental structure of P. densiflora f. multicaulis can be selected. In addition, we could find genetic differences according to the location of female strobili. Future studies such as the functional study of the genes found in this study and the development of the markers according to the results of SNPs molecular markers will be necessary.
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Keyword : Pinus densiflora f. multicaulis Uyeki, bisexual strobilus, conelets, RNA-seq, SNP
Abbreviations :
FS : female strobilus, seed cone, ovulate strobilus MS : male strobilus, pollen cone, staminate strobilus FSS : seed cone at the side aspect
BS : bisexual strobilus AC : apical cone SC : side cone
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Supplemental data
S1. Desctiption of mapped transcripts with gene functional data base sets of Arabidopsis thailiana and Pinus sylvestris L.
DB ID Description
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